Forty percent of children with acute myeloid leukemia (AML) who achieve remission subsequently experience frank relapse of their disease and their ultimate prognosis is typically poor. Unfortunately, such disease recurrences cannot be accurately predicted with current laboratory techniques. With the advent of multidimensional flow cytometry, rare leukemic cells can be identified when they are undetectable by conventional light microscopy. Using aberrant cell surface antigen expression patterns observed in AML to distinguish these cells from normal bone marrow, we have identified patients harboring detectable residual leukemic cells during complete remission. In a prospective, cooperative trial of 252 pediatric AML patients, children harboring detectable leukemia in remission were four times more likely to experience relapse and three times more likely to die than those without detectable leukemia. In fact, flow cytometric evidence of leukemia after induction therapy emerged as the most powerful independent prognostic factor associated with poor outcome. Overall survival at 3 years was 41% vs. 69% for patients with and without occult leukemia, respectively (P=0.006). These findings establish rationale for intensifying post-remission therapy in high-risk patients. Prediction of relapse at a time of minimal disease burden would be of significant clinical value, as it would allow early intervention with a more aggressive form of therapy (i.e., allogeneic marrow transplant) to achieve an improved overall cure rate. Moreover, as the flow cytometric assay improves, it may become possible to safely decrease the dose and intensity of chemotherapy given to low-risk patients as a means of limiting the short- and long-term health consequences associated with aggressive treatment. Our prospective studies have demonstrated that the multidimensional flow cytometric assay is highly specific for leukemic cells, in fact, almost every patient with residual leukemia detectable by the MDF assay who did not receive a subsequent allogeneic transplant subsequently relapsed. However, a significant portion of remission patients in whom no leukemia was detected by this assay also experienced disease recurrence. For this reason, we now seek to evaluate several means of increasing the sensitivity of the assay. Specifically we plan to: (1) study an increased number of cells per assay tube, (2) evaluate various novel myeloid and lymphoid antibody combinations that should allow greater distinction between normal cells and their malignant counterparts, and (3) increase the number of antibody combinations per tube. With these enhancements and modifications, we believe we will be able to accurately predict relapse in more than 80% of children with AML who achieve remission.